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Abstract Background Endoclita signifer causes severe damage to eucalyptus plantations, and the larvae transfer to and damage eucalyptus accurately in mixed forests, suggesting that the larval olfactory system contributes to host selection. The olfactory proteins in the head and tegument of E. signifer larvae were previously identified. To identify the relationship between olfactory protein expression in the larval head the larvae head and the developmental expression dynamics, and its functions in further recognition of plant volatiles, the head transcriptomes of two instar larvae and the expression profiles of olfactory proteins in the instars after exposure to volatiles were studied. Results Eight odorant-binding proteins, six chemosensory proteins, three odorant receptors, three gustatory receptors, and 18 ionotropic receptors were identified. Half of the olfactory proteins were the most highly expressed in the young (5th) larval head, and EsigGOBP2, EsigGOBP4, EsigGOBP5, EsigCSP1, EsigCSP3, EsigGR1 and EsigGR3 were highly expressed and showed a specific expression pattern. In addition, after exposure to o-cymene, α-phellandrene, n-butyl ether, and 4-ethylacetophenone, EsigGR3 was downregulated significantly, and exposure to n-butyl ether caused EsigGR1 to be downregulated significantly. Conclusions Seven specific olfactory proteins may be important genes in larval olfactory recognition. Furthermore, based on the receptors that were downregulated after exposure to volatiles and the previous electrophysiological activity in the third larvae, we speculated that the ligand of EsigGR1 was n-butyl ether, and the ligands of the newly identified EsigGR3 are all electrophysiologically active compounds, which demonstrated host recognition in the third larvae of E. signifer. These results provide a way to find key plant volatiles recognized by the key olfactory proteins as new targets for pest control. Graphical Abstract |